1. Field of the Invention
The present invention relates generally to the delivery of biologically active molecules, such as peptides, polypeptides and nucleic acids, into the interior of cells. Specifically, the delivery is accomplished by administering to the cells a complex comprising the molecule linked to a signal peptide. Such delivery can be utilized for many purposes such as peptide therapy, antisense oligonucleotide therapy and gene transfer.
2. Background Art
Peptides have been developed for many therapeutic uses. For example, diseases currently targeted by new peptide drugs include heart conditions, cancers, endocrine disorders, neurological defects, respiratory conditions, allergies and autoimmune diseases. Although the manufacture of known therapeutic peptides can be achieved by known methods, i.e., classic synthetic techniques or recombinant genetic engineering, delivery of the peptides into a cell has remained problematic, since they cannot readily cross biological membranes to enter cells. Thus, current methods include permeabilization of the cell membrane or microinjection into the cell. Both of these methods have serious drawbacks. Permeabilization of cells, e.g., by saponin, bacterial toxins, calcium phosphate, electroporation, etc., can only be practically useful for ex vivo methods, and these methods cause damage to the cells. Microinjection requires highly skilled technicians (thus limiting its use to a laboratory setting), it physically damages the cells, and it has only limited applications as it cannot be used to treat, for example, a mass of cells or an entire tissue, because one cannot feasibly inject large numbers of cells.
Similarly, delivery of nucleic acids has been problematic. Methods currently employed include the permeabilization described above, with the above-described drawbacks, as well as vector-based delivery, such as with viral vectors, and liposome-mediated delivery. However, viral vectors can present additional risks to a patient, and liposome techniques have not achieved satisfactorily high levels of delivery into cells.
Signal peptide sequences,.sup.1 which share the common motif of hydrophobicity, mediate translocation of most intracellular secretory proteins across mammalian endoplasmic reticulum (ER) and prokaryotic plasma membranes through the putative protein-conducting channels..sup.2-11 Alternative models for secretory protein transport also support a role for the signal sequence in targeting proteins to membranes..sup.12-15
Several types of signal sequence-mediated inside-out membrane translocation pathways have been proposed. The major model implies that the proteins are transported across membranes through a hydrophilic protein-conducting channel formed by a number of membrane proteins..sup.2-11 In eukaryotes, newly synthesized proteins in the cytoplasm are targeted to the ER membrane by signal sequences that are recognized generally by the signal recognition particle (SRP) and its ER membrane receptors. This targeting step is followed by the actual transfer of protein across the ER membrane and out of the cell through the putative protein-conducting channel (for recent reviews, see references 2-5). In bacteria, the transport of most proteins across the cytoplasmic membrane also requires a similar protein-conducting channel..sup.7-11 On the other hand, signal peptides can interact strongly with lipids, supporting the proposal that the transport of some secretory proteins across cellular membranes may occur directly through the lipid bilayer in the absence of any proteinaceous channels..sup.14-15
Thus, though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. This problem affects a wide variety of therapies. The solution of this problem would greatly expand treatments of diseases for which delivery of a therapeutic molecule would be beneficial. The present invention solves this long-felt, broad spectrum problem by providing a method of importing a biologically active molecule into a cell using mechanisms naturally occurring in cells and thus avoiding damaging the target cells. Additionally, the present method can be used to import molecules into large numbers of cells, including organs. Thus, this versatile inventive method can be employed in numerous treatments of diseases and disorders.